| Abstract [eng] |
The yeasts Saccharomyces cerevisiae are safe organisms for humans. These yeasts have been used in the fermentation of various baked goods and beverages for thousands of years (Landry et al., 2006). A 2007 study of the human microbiome found that S. cerevisiae is also part of the natural microflora of healthy humans (Nash et al., 2017). This yeast is important and widely used in food (Legras et al., 2007), medicine, pharmaceutical industry and biofuel production (Türker, 2014). Saccharomyces cerevisiae is one of the model eukaryotic organisms that provides extensive knowledge of cellular processes (Landry et al., 2006). Most strains of Saccharomyces cerevisiae carry the L-A virus. These are 4.6 kb dgRNA viruses that are stably maintained in yeast cells and do not adversely affect their growth. L-A-1 capsid proteins have been found to form self-assembling VLPs (Fujimura et al., 1992). Such pseudovirion particles are applicable in the medical industry. One of the uses of VLPs is to adapt them to the development of various vaccines or delivery systems (Fuenmayor, Gòdia, & Cervera, 2017). The aim of this scientific work was to evaluate the possibility of applying L-A-1 virus VLPs as a nanocarriers. In particular, purification of VLP from the strains of S. cerevisiae BYΔLA, BYΔLA GagWT, BY4741 GagWT, and BY4741 GagΔ using the sucrose cushion method was used to confirm that L-A-1 virus components do not adversely affect the viability of the human cancer cell line HCT116. The highest levels of L-A-1 virus VLPs were found to be produced in the BY4741 ΔPOR1 GagWT strain, and the most efficient VLPs purification method was using PEG4000. Purification method using PEG4000 is 10 times more efficient than purification by sucrose gradient ultracentrifugation according to purified Gag protein concentration. Transfection capacity of purified viral particles showed that at low Dox concentrations (20 and 50 ng / ml), the cytotoxic effect of Dox on HCT116 cells is enhanced when the cells are co-cultured with L-A-1 virus VLP. Therefore, these particles may potentially improve the uptake of Dox into HCT116 cells at low concentrations (20 and 50 ng / ml), thus expanding the effective concentration spectrum of Dox. Monitoring of Dox fluorescence in exposed HCT116 cells also suggests that Dox enters cells efficiently when co-cultured with L-A-1 virus VLPs. |
